The overall goal of this proposal is to develop methods for characterizing stable base-pairing interactions between complementary RNA molecules in vivo. These methods will be used to characterize the presence of double-stranded RNA within in mammalian cells in order to analyze mechanisms for antisense regulation. Although antisense regulation has been well-documented and mechanistically explored in several prokaryotic systems relatively little is known regarding the mechanism by which antisense RNAs alter the expression of complementary transcripts in eukaryotes. However, there is accumulating evidence that many regions of the mammalian genome expressed overlapping that are transcribed from both strands of the chromosome. In some instances this transcription may give rise to antisense regulation in which expression of a target transcript is repressed by base pairing with complementary antisense RNA present. The proposed study has two specific goals: (1) to develop methods for the isolation, identification and characterization of doublestranded RNA formed in vivo; and (2) to prepare libraries of amplified cDNAs from isolated dsRNA that will be screened using either random or targeted strategies. A key feature of this strategy is that single-stranded RNAs will be modified prior to isolation to prevent adventitious annealing. To facilitate identification of condition that are optimal for isolation of native double-stranded RNA, a synthetic RNA hairpin will express in transfected embryonic carcinoma cells to serve as a target sequence in Specific Aim 1. Finally, libraries will be screened for complementary overlapping sequences using both gene specific probes and high throughput methods to characterize the population of stable double-stranded RNAs. The results of these studies will provide insight into the formation and stability of RNA-RNA base-paired interactions in vivo, and the methods and information needed for further studies of the functional roles of such interactions.